High-throughput stem cell assay of hematopoietic stem and progenitor cell proliferation

ABSTRACT

The present invention relates generally to high-throughput assay methods that determine the proliferative status of hematopoietic stem and progenitor cells. The present invention further relates to high-throughput assays for screening compounds that modulate the growth of hematopoietic stem and progenitor cells and for identifying subpopulations thereof that are suitable for transplantation. The assay of the present invention is particularly useful for quality control and monitoring of the growth potential in the stem cell transplant setting and would provide improved control over the reconstitution phase of transplanted cells.

PRIORITY CLAIM The government has certain rights in the invention.

[0001] The applicant claims the benefit of the filing date of U.S.provisional application, Serial No. 60/264,796, filed Jan. 29, 2000, thecontents of which are also hereby incorporated by reference in theirentireties.

[0002] This invention was made with government support under SmallBusiness Innobation Research (SBIR) grant number 1 R43CA93244-02 awardedby the NIH.

FIELD OF THE INVENTION

[0003] The present invention relates generally to high-throughput assaymethods that determine the proliferative status of hematopoietic stemand progenitor cells. The present invention further relates tohigh-throughput assays for screening compounds that modulate theproliferation of hematopoietic stem and progenitor cells and foridentifying subpopulations thereof that are suitable fortransplantation.

BACKGROUND

[0004] Two major concerns in drug development are the need to predictthe efficacy and safety of a potential drug candidate before clinicaltrials are initiated, and how to predict which individual cancer casesare going to respond to a particular treatment. During the developmentprocess, therefore, drug candidates are typically batch tested on avariety of cell lines. Those that seem to have the desired effect on thecell lines are then tested in animals during the preclinical phase.Neither process, however, replicates the true clinical situation. As aresult, patients in clinical trials often have to endure unpleasant andsometimes harmful effects because neither differential sensitivity norpatient variability was adequately considered during the drugdevelopmental phases.

[0005] To overcome these deficiencies, in vitro cell-based assays thatdemonstrate a high degree of predictability during different phases ofdrug development are needed. In addition, there is a need for highthroughput assays for screening large numbers of potential drugcandidates produced during the discovery phase of drug development andallow those demonstrating promise to continue further in research anddevelopment. It therefore follows that assays based on primary cellsexhibiting a high degree of predictability, coupled with ahigh-throughput component, could have a significant impact and value onthe drug development process.

[0006] The hematopoietic system is one of five continuouslyproliferating systems of the body, the others being the epithelialmucosa of the gastrointestinal tract, the dermis of the skin, the germcells of the reproductive organs and the epithelium of the eye cornea.All five proliferating systems share common characteristics, the mostimportant being that a small population of stem cells maintains thecontinuous production of mature end cells. They all possess the samestructural organization of four basic compartments, namely the stemcell, amplification and differentiation, maturation and mature cellcompartments.

[0007] The hematopoietic system, however, is unique in several ways. Itis the only system capable of producing at least eight functionallydifferent cell lineages from a single pluripotent stem cell. Assays areavailable that allow the differential effect of drugs on the variouslympho-hematopoietic lineages to be examined. Second, the site of cellproduction changes during ontological development. This helps indifferential sensitivity testing. Third, the site of production in theadult is the bone marrow, which is a significantly different tissue fromthe functional site of the peripheral circulation. Fourth, compared withother proliferating systems, and almost all other systems of the body,adult hematopoietic stem and progenitor cells are readily accessible.

[0008] Hematopoietic stem and progenator cell lineages can be used tomeasure parameters that would normally be inaccessible. For example, thefunctional site of hematopoiesis is the circulation and mature end cellscan be readily obtained to measure red and white blood cell counts,differential counts and other end stage blood parameters. Theseparameters are conventionally used in preclinical drug testing and formthe basis of the National Cancer Institute (NCI) guidelines forhemotoxicity testing during clinical trials. However, these parametershave little if any predictive value as to, for example, the cytotoxiceffect of therapeutic compounds on primitive hematopoietic cells or thestem cells of other proliferating tissues.

[0009] Besides the mature red and white blood cells, the peripheralblood also contains circulating populations of stem and progenitor cellsthat can be isolated and used for hematopoietic status monitoring andhemotoxicity testing. The so-called granulocyte-macrophagecolony-forming cell (GM-CFC) assay and the enumeration of CD34⁺ cells(stem and early progenitor cells) currently form the basis of qualitycontrol for hematopoietic stem cell transplantation.

[0010] The widespread use of in vitro hematopoietic assays was initiatedwhen soluble factors released by fibroblasts were shown to be capable ofstimulating cells to form granulocyte-macrophage colonies in soft agar(Bradley & Metcalf, Aust. J Exp. Biol. Med. 44, 287-287 (1987); Pluznik& Sachs, Exp. Cell Res. 43, 553-553 (1966)). Colony forming assays(CFAs) for erythropoietic progenitor cells (McLeod et al., Blood 44,617-534 (1974); Iscove et al., J. Cell Phyisol. 2-23 (1974); Axelrad etal., Haemopiesis in Culture 226-223 (1974)) and other hematopoieticlineages were also developed. The use of cytotoxic drugs such as5-fluorouracil (Hodgson et al., Exp. Hemat. 10, 26-36 (1982) and Int. J.Cell Cloning 1, 49-56 (1983)) and hydroxyurea (Rosendaal et al., Nature264, 68-69 (1976)) allowed the hierarchy within the stem cellcompartment to be elucidated and in vitro assays for primitive stem cellpopulations to be developed (Ploemacher et al., Blood 78, 2527-2536(1991); Sutherland et al., Blood 72, 104a (1988)).

[0011] In vivo, an insult at the stem or early progenitor cell levelrequires a certain amount of time for the effect to be detected at theperipheral blood level. The effect may not be observed for weeks, oreven months. This does not provide a high level of predictability and iswhy end stage cell parameters cannot be used to predict the effect of anagent. By the time the effect is observed, adverse reactions by thepatient have already occurred.

[0012] In vitro colony-forming assays based on stem or progenitor cells,on the other hand, can fulfill the requirements of prediction andsensitivity because they detect the effect of the insult before it isobserved in the circulation. Colony-forming assays for leukemic cellsare also available. In these classic assays, the more primitive the cellto be detected, the longer it takes to detect its progeny in the form ofa colony. The proliferative potential of the cells being analyzed, andtheir ability to be stimulated by growth factors in vitro are essentialfor these assays. This dependency on the amplification compartmentinherent in the hematopoietic system is often overlooked and withoutthis component colony-forming assays in general, and especiallypredictive hemotoxicity testing, could not be performed.

[0013] Under steady-state conditions, the proliferative status ofprimitive stem cells is considered to be quiescent, while the proportionof cells in cell cycle increases with stem cell maturity. Once the stemcell has become determined with respect to a cell lineage, it enters theamplification compartment for producing the large and constant number ofmature cells. With entry into the cell cycle, however, the cell becomesvulnerable to exogenous agents including the cytotoxic drugs typicallyused in oncology. Thus, the GM-CFC assay, for example, has been used topredict myelosuppression (Prieto, P., Sci. Total Environ. 247, 349-354(2000)). The predictive quality of this assay, has been proven byvalidation studies with alkylating agents (Parchment et al., Toxicol.Pathol. 21, 241-250 (1993)). Additionally, however, if the maximumtolerated drug concentration for hematopoietic cells can be predicted,hemotoxicity studies would play an important role in drug discoverysince it would be a therapeutic index-based assay (Parchment et al.,Ann. Oncol. 9, 357-364 (1998)).

[0014] In the case of cytotoxic drug testing, the target cells have tobe in cell cycle. For any drug that relies on cell proliferation, thetissues most affected or damaged by toxicity are those actively engagedin cell proliferation, which includes the bone marrow and thegastrointestinal tract. It therefore follows that hemotoxicity testingcould also usefully be extrapolated to, and predictive for, the effectsof a potential drug on other proliferating tissues.

[0015] Toxicity in general, and hemotoxicity in particular, can also becorrelated with the time of drug administration. The therapeutic indexof a drug, and hence its toxicity, is dependent, in part on thecircadian variation in the hematopoietic cell division of rodents(Laerum, O. D., Exp. Hematol. 23, 1145-1147 (1995); Aardal et al., Exp.Hemtol., 11, 792-801 (1993); Aardal, Exp. Hematol. 12, 61-67 (1984);Wood et al., Exp. Hematol., 26, 523-533 (1998)), dogs (Haurie et al.,Exp. Hematol. 27, 1139-1148 (1999); Abkowitz et al., Exp. Hematol. 16,941-945 (1988)) and in humans (Abrahamsen et al., Eur. J Haematol. 58,333-345 (1997); Baudoux et al., Bone Marrow Transplant 22 (Suppl. 1) S12 (1998); Carulli et al., Hematologica 85, 447-448 (2000)). Similarly,cells of the gut mucosa (Schering et al., Anat. Rec. 191, 479-486(1978)), Stenn & Paus, Exp. Dermatol. 8, 229-233 (1999); Zanello et al.,J. Invest. Dermatol. 115, 757-760 (2000)) and the corneal epithelium ofthe eye (Schening et al., Anat. Rec. 191, 479-486 (1978)) exhibitcircadian organization. For human bone marrow and gastrointestinaltissues, for example, S-phase DNA synthesis preferentially occurs in themorning hours rather than in the evening or nighttime hours. Thisimplies cytotoxic agents might be less toxic and exhibit high efficacyif given at a time when the proliferative status of the cells is at anadir in these tissues.

[0016] For toxicity testing, large numbers of comparative samples areneeded, thereby making the enumeration of manual CFAs for this purposeimpractical. CFAs also suffer from a lack of standardized colonyenumeration procedures, and the subjectivity and high degree ofexpertise of the personnel and the time required for accurateenumeration of the colonies. The long culture periods required tovisualize the proliferative potential of different cell populations isalso a disadvantage. However, the culture period is an inherent propertyof the cell population and cannot be changed.

[0017] Conventional cell proliferation assays have measured either³H-thymidine or 5-bromo-deoxyuridine (BrdU) incorporation. The BrdUassay can use microscopy, flow cytometry or absorbance. Colorimetrictetrazolium compounds, in particular3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT),(Mosmann, J. Immunol. Meth. 65, 666 (1983)), have also been used.Horowitz and King, J. Immunol. Meth. 244, 49-58 (2000)) developed amulti-well, murine colony-forming assay in soft agar whereby theenumeration of cell proliferation or inhibition was measured using theMTT calorimetric method. Results were equivalent to the colony-formingassay. The number of target cells was reduced to 1.25×10⁴ cells/ml, butonly studied granulocyte/macrophage progenitor cells were tested and notstem cells, erythropoietic or megakaryopoietic progenitor cells.However, it is also desirable to have an assay system that canaccommodate the complete range of target cell populations that can becultured and subjected to drug-induced hemotoxicity effects.

[0018] Hematological malignancies rank 5th and 6th in the cause ofdeaths for men and women respectively and use of stem cell transplantsusing peripheral blood, bone marrow and umbilical cord blood haveincreased dramatically. Reconstitution of the patient after atransplant, however, usually occurs in about 14 days, which is the sametime required for the conventional, manual, CFA to detect the growthpotential of transplanted cells. Therefore, the usefulness of the GM-CFCassay as an indicator and quality control measure for the growthpotential of the transplantable cells is limited. Reliance is oftenplaced on measuring the number of CD34+ cells by flow cytometery, eventhough this provides no information as to the cell growth potential.Therefore, there is a need for a sensitive, rapid and cost-effectiveassay that can be used as an indicator for hematopoietic engraftment andreconstitution potential. The patient would benefit significantlybecause, if engraftment and reconstitution of the lympho-hematopoieticsystem does not occur after transplantation, the physician can rapidlydetect this rejection and proceed with a second transplant, offeringreduced financial implications in lower hospitalization and medicationcosts and improved patient comfort and recovery.

[0019] These and other objectives and advantages of the invention willbecome fully apparent from the description and claims that follow or maybe learned by the practice of the invention.

SUMMARY OF THE INVENTION

[0020] Briefly described, the present invention relates generally tohigh-throughput assay methods that determine the proliferative status ofhematopoietic stem and progenitor cells. The present invention furtherrelates to high-throughput assays for screening compounds that modulatethe growth of hematopoietic stem and progenitor cells and foridentifying subpopulations thereof that are suitable fortransplantation. The assay of the present invention is particularlyuseful for quality control and monitoring of the growth potential in thestem cell transplant setting and would provide improved control over thereconstitution phase of transplanted cells.

[0021] The present invention addresses the need for rapid assays thatwill determine the proliferative status of isolated hematopoietic stemand progenitor cells and of subpopulations of differentiated cellsthereof.

[0022] One aspect of the present invention provides a high-throughputassay method useful for rapidly determining the proliferative status ofa population of primitive hematopoietic cells as a function of the ATPcontent of the cells, the method comprising incubating a primitivehematopoietic cell population in a cell growth medium having aconcentration of fetal bovine serum of between 0% and about 30%, aconcentration of methyl cellulose between about 0.4% and about 0.7%, andin an atmosphere having less than about 7.5% oxygen. The cell populationis contacted with a reagent capable of generating luminescence in thepresence of ATP and the luminescence generated by the reagent isdetected. The level of luminescence indicates the amount of ATP in thecell population, wherein the amount of ATP indicates the proliferativestatus of the primitive hematopoietic cells.

[0023] One embodiment of the method of the present invention furthercomprises the step of contacting the primitive hematopoietic cellpopulation with at least one cytokine and optionally may furthercomprising the step of generating a cell population substantiallyenriched in hematopoietic stem cells.

[0024] In another embodiment of the method of the present invention, thecell population is substantially enriched in at least one hematopoieticprogenitor cell lineage.

[0025] Another aspect of the present invention is a high-throughputassay method for rapidly identifying a population of primitivehematopoietic cells having a proliferative status suitable fortransplantation into a patient. The method of the present invention,therefore, may comprise incubating a primitive hematopoietic cellpopulation in a cell growth medium having a concentration of fetalbovine serum between 0% and about 30%, a concentration of methylcellulose between about 0.4% and about 0.7%, and in an atmosphere havingless than about 7.5% oxygen. The primitive hematopoietic cell populationis contacted with at least one cytokine, typically before the incubationof the cells. Thereafter, the cell population is contacted with areagent capable of generating luminescence in the presence of ATP. Theluminescence thereby generated indicates the proliferative status of theprimitive hematopoietic cells, which in turn indicates the suitabilityof the cell population for transplantation into a recipient patient.

[0026] Yet another aspect of the present invention is a high-throughputassay method for rapidly identifying a compound capable of modulatingthe proliferative status of a population of primitive hematopoieticcells. In this aspect of the present invention, a first target cellpopulation comprising primitive hematopoietic cells is incubated in cella growth medium having a concentration of fetal bovine serum between 0%and about 30%, a concentration of methyl cellulose between about 0.4%and about 0.7%, and in an atmosphere having less than about 7.5% oxygen.The method further comprises providing a plurality of second targetprimitive hematopoietic cell populations, contacting the first andsecond primitive hematopoietic cell populations with at least onecytokine before incubating the cell cultures, contacting the first andsecond target cell populations with at least one test compound,contacting the target cell populations with a reagent capable ofgenerating luminescence in the presence of ATP. The luminescencegenerated is detected by the reagent contacting the target cellpopulations, the level of luminescence indicating the proliferativestatus of the primitive hematopoietic cells. The proliferative status ofthe plurality of the second target cell populations with theproliferative status of the first target population of primitivehematopoietic cells not in contact with the test compound, therebyidentifying a test compound capable of modulating the proliferativestatus of a target cell population.

[0027] Additional objects and aspects of the present invention willbecome more apparent upon review of the detailed description set forthbelow when taken in conjunction with the accompanying figures, which arebriefly described as follows.

BRIEF DESCRIPTION OF THE FIGURES

[0028] FIGS. 1A-4B illustrate the correlation between the initial platedcell concentration (0.25, 0.5, 0.75, 1, 1.5 2×10⁵/well) and the mean(FIGS. 1A, 2A, 3A and 4A respectively) or sum (FIGS. 1B, 2B, 3B, and 4Brespectively) of relative luminescence units (RLU) measured at 4 days(FIGS. 1A and 1B), 7 days (FIGS. 2A and 2B), 10 days (FIGS. 3A and 3B)and 14 days (FIGS. 4A and 4B) after culture initiation, as a function ofthe integration time and/or gain of the plate reader. In FIGS. 1A-4B thevalue 2000 represents an integration time of 200 ms. “Max” representsthe maximum integration time. The values 200, 215, 225 or 250 representthe gains that were used with the respective integration times.

[0029] FIGS. 5A-5C illustrate histograms showing the number of cellclusters counted manually per well and the relative luminescence units(RLU) per well at day 7 (FIG. 5A), day 10 (FIG. 5B) and day 14 (FIG. 5C)of incubation.

[0030] FIGS. 6A-6C graphically illustrate the lack of correlationbetween cell cluster counts per well and the relative luminescence units(RLU) per well on day 7 (FIG. 6A), day 10 (FIG. 6B) and day 14 (FIG. 6C)of culture incubation.

[0031] FIGS. 7A-7C show the direct correlation between the sum, or mean,of the cell cluster counts with the sum or mean of the relativeluminescence units (RLU) measured on day 7 (FIG. 7A), day 10 (FIG. 7B)and day 14 (FIG. 7C) of culture incubation.

[0032] FIGS. 8A-8C show the correlation between cell concentration, sumof the replicate cell clusters and mean of the replicate cell clusterson day 7 (FIG. 8A), day 10 (FIG. 8B) and day 14 (FIG. 8C) of cultureincubation.

[0033] FIGS. 9A-9C show the correlation between the original manual4-well assay and the 96-well assay, method of the present invention. Theresults were plotted as either the sum or mean of the replicatesobtained on day 7 (FIG. 9A), day 10 (FIG. 9B) and day 14 (FIG. 9C) ofculture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] A full and enabling disclosure of the present invention,including the best mode known to the inventor of carrying out theinvention, is set forth more particularly in the remainder of thespecification, including reference to the Examples. This description ismade for the purpose of illustrating the general principles of theinvention and should not be taken in the limiting sense.

[0035] The methods of the present invention provides high-throughputassays for detecting and measuring the proliferative status ofpopulations of primitive hematopoietic stem and progenitor cells, andcell lineages derived therefrom.

[0036] The methods of the present invention are especially useful whenapplied to populations of primitive hematopoietic cells includingprimary cells isolated from peripheral blood cells and bone marrow cellsand hematopoietic stem and progenitor cells. The methods of the presentinvention, however, may be applied to any population of proliferatingcells, including cells isolated from tissues and solid tumors.

[0037] The methods of the present invention can also be used todistinguish subpopulations of cells that may differ in the response tocytotoxic inhibitors, or activators such as cytokines The methods may beused to optimize the inhibitors to achieve maximum efficacy against asubpopulation of proliferating cells. An optimized dose, determined froman isolated small sample of the cell population of a patient, may beadministered to the proliferating cells in vivo, wherein the optimizeddose may be administered systemically to the human or animal patienthaving the proliferating subpopulation of cells, thereby reducing thelikelihood of potentially harmful side-effects to the recipient patient.

[0038] The high-throughput assay methods of the present invention mayalso be used to determine the proliferative status of a population ofhematopoietic stem or progenitor cells to determine their suitabilityand acceptability for transplantation into a recipient animal or humanpatient.

[0039] Definitions

[0040] The term “animal” as used herein refers to any vertebrate animalother than a human having a population of cells wherein at least onesubpopulation of the cells may be proliferating or induced toproliferate. The term “animal” as used herein also refers to mammalsincluding, but not limited to, bovine, ovine, porcine, equine, canine,feline species, non-human primates including apes and monkies, rodentssuch as rat and mouse, and lagomorphs such as rabbit and hare.

[0041] The term “tissue” as used herein refers to a group or collectionof similar cells and their intercellular matrix that act together in theperformance of a particular function. The primary tissues areepithelial, connective (including blood), skeletal, muscular, glandularand nervous.

[0042] The term “cell” or “cells” as used herein refers to any cellpopulation of a solid or non-solid tissue including, but not limited to,a peripheral blood cell population, bone marrow cell population, aleukemic cell line population and a primary leukemic cell linepopulation or a blood stem cell population. The cells may behematopoietic cells, including bone marrow, umbilical cord blood, fetalliver cells, yolk sac and differentiating embryonic stem cells ordifferentiating primordial germ cells or embryonic germ cells. The cellsmay be a primary cell line population including, but not limited to, aleukemic cell line. Examples of leukemic cell lines include, but are notlimited to, an acute lymphocytic leukemia, an acute myeloid leukemia, achronic lymphocytic leukemia, a chronic myeloid leukemia and a pre-Bacute lymphocytic leukemia. Such cell lines include, but are not limitedto, acute myelogenous leukemia, acute T-cell leukemia, acutelymphoblastic leukemia, chronic myeloid leukemia, acute monocyticleukemia and B-cell leukemia. The term “target cell population” as usedherein refers to any cell population, especially hematopoietic stem andprogenitor cells, or subpopulations thereof, that may be contacted witha test compound, wherein the test compound may modulate theproliferation of the cells in a positive or a negative directiondepending upon the compound and the target cell population.

[0043] The term “cell line” refers to cells that are harvested from ahuman or animal adult or fetal tissue, including blood and cultured invitro, including primary cell lines, finite cell lines, continuous celllines, and transformed cell lines.

[0044] The term “cell lineage” as used herein refers to a cell linederived from a progenitor or stem cell, including, but not limited to ahematopoietic stem or progenitor cell.

[0045] The term “cell cycle” as used herein refers to the cycle ofstages in the replication of a eukaryotic cell. The cycle comprises thefour stages G1, S, G2 and M, wherein the S phase is that portion of thecycle wherein the nucleic acid of the cell is replicated. Thus, a cellidentified as being in the S-phase of the cell cycle is also identifiedas being a proliferating cell.

[0046] The term “proliferative status” as used herein refers to whethera population of hematopoietic stem or progenitor cells, or asubpopulation thereof, are dividing and thereby increasing in number, inthe quiescent state, or whether the cells are not proliferating, dyingor undergoing apoptosis.

[0047] The terms “modulating the proliferative status” or “modulatingthe proliferation” as used herein refers to the ability of a compound toalter the proliferation rate of a population of hematopoietic stem orprogenitor cells A compound may be toxic wherein the proliferation ofthe cells is slowed or halted, or the proliferation may be enhanced suchas, for example, by the addition to the cells of a cytokine or growthfactor.

[0048] The term “quiescent” refers to cells that are not activelyproliferating by means of the mitotic cell cycle. Quiescent cells (whichinclude cells in which quiescence has been induced as well as thosecells which are naturally quiescent, such as certain fullydifferentiated cells) are generally regarded as not being in any of thefour phases G1, S, G2 and M of the cell cycle; they are usuallydescribed as being in a G0 state, so as to indicate that they would notnormally progress through the cycle. Cultured cells can be induced toenter the quiescent state by various methods including chemicaltreatments, nutrient deprivation, growth inhibition or manipulation ofgene expression, and induced to exit therefrom by contacting the cellswith cytokines or growth factors.

[0049] The term “primary cell” refers to cells obtained directly from ahuman or animal adult or fetal tissue, including blood. The “primarycells” or “cell lines” may also be derived from a solid tumor or tissue,that may or may not include a hematopoietic cell population, and can besuspended in a support medium. The primary cells may comprise a primarycell line.

[0050] The term “primitive hematopoietic cell” as used herein refers toany stem, progenitor or precursor cell that may be induced todifferentiate and/or proliferate to form a population of hematopoieticcells.

[0051] The term “hematopoietic stem cells” as used herein refers topluripotent stem cells or lymphoid or myeloid stem cells that, uponexposure to an appropriate cytokine or plurality of cytokines, mayeither differentiate into a progenitor cell of a lymphoid or myeloidcell lineage or proliferate as a stem cell population without furtherdifferentiation having been initiated. “Hematopoietic stem cells”include, but are not limited to, colony-forming cell-blast (CFC-blast),high proliferative potential colony forming cell (HPP-CFC) andcolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte(CFU-GEMM) cells.

[0052] The terms “progenitor” and “progenitor cell” as used herein referto primitive hematopoietic cells that have differentiated to adevelopmental stage that, when the cells are further exposed to acytokine or a group of cytokines, will differentiate further to ahematopoietic cell lineage. “Progenitors” and “progenitor cells” as usedherein also include “precursor” cells that are derived from some typesof progenitor cells and are the immediate precursor cells of some maturedifferentiated hematopoietic cells. The terms “progenitor”, and“progenitor cell” as used herein include, but are not limited to,granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocytecolony-forming cell (CFC-mega), burst-forming unit erythroid (BFU-E),colony-forming cell-megakaryocyte (CFC-Mega), B cell colony-forming cell(B-CFC) and T cell colony-forming cell (T-CFC). Precursor cells”include, but are not limited to, colony-forming unit-erythroid (CFU-E),granulocyte colony forming cell (G-CFC), colony-forming cell-basophil(CFC-Bas), colony-forming cell-eosinophil (CFC-Eo) and macrophagecolony-forming cell (M-CFC) cells.

[0053] The term “cytokine” as used herein refers to any cytokine orgrowth factor that can induce the differentiation of a hematopoieticstem cell to a hematopoietic progenitor or precursor cell and/or inducethe proliferation thereof. Suitable cytokines for use in the presentinvention include, but are not limited to, erythropoietin,granulocyte-macrophage colony stimulating factor, granulocyte colonystimulating factor, macrophage colony stimulating factor,thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin. Theterm “cytokine” as used herein further refers to any natural cytokine orgrowth factor as isolated from an animal or human tissue, and anyfragment or derivative thereof that retains biological activity of theoriginal parent cytokine. The cytokine or growth factor may further be arecombinant cytokine or a growth factor such as, for example,recombinant insulin. The term “cytokine” as used herein further includesspecies-specific cytokines that while belonging to a structurally andfunctionally related group of cytokines, will have biological activityrestricted to one animal species or group of taxonomically relatedspecies, or have reduced biological effect in other species.

[0054] The terms “cell surface antigen” and “cell surface marker” asused herein may be any antigenic structure on the surface of a cell. Thecell surface antigen may be, but is not limited to, a tumor associatedantigen, a growth factor receptor, a viral-encoded surface-expressedantigen, an antigen encoded by an oncogene product, a surface epitope, amembrane protein which mediates a classical or atypical multi-drugresistance, an antigen which mediates a tumorigenic phenotype, anantigen which mediates a metastatic phenotype, an antigen whichsuppresses a tumorigenic phenotype, an antigen which suppresses ametastatic phenotype, an antigen which is recognized by a specificimmunological effector cell such as a T-cell, and an antigen that isrecognized by a non-specific immunological effector cell such as amacrophage cell or a natural killer cell. Examples of “cell surfaceantigens” within the scope of the present invention include, but are notlimited to, CD3, CD4, CD8, CD34, CD90 (Thy-1) antigen, CD117, CD38,CD56, CD61, CD41, glycophorin A and HLA-DR, AC133 defining a subset ofCD34⁺ cells, CD19, and HLA-DR. Cell surface molecules may also includecarbohydrates, proteins, lipoproteins or any other molecules orcombinations thereof, that may be detected by selectively binding to aligand or labeled molecule and by methods such as, but not limited to,flow cytometry.

[0055] The term “cell surface indicator” as used herein refers to acompound or a plurality of compounds that will bind to a cell surfaceantigen directly or indirectly, and thereby selectively indicate thepresence of the cell surface antigen. Suitable “cell surface indicators”include, but are not limited to, cell surface antigen-specificmonoclonal or polyclonal antibodies, or derivatives or combinationsthereof, and which may be directly or indirectly linked to a signalingmoiety. The “cell surface indicator” may be a ligand that can bind tothe cell surface antigen, wherein the ligand may be a protein, peptide,carbohydrate, lipid or nucleic acid that is directly or indirectlylinked to a signaling moiety.

[0056] The term “flow cytometer” as used herein refers to any devicethat will irradiate a particle suspended in a fluid medium with light ata first wavelength, and is capable of detecting a light at the same or adifferent wavelength, wherein the detected light indicates the presenceof a cell or an indicator thereon. The “flow cytometer” may be coupledto a cell sorter that is capable of isolating the particle or cell fromother particles or cells not emitting the second light.

[0057] The term “reagent capable of generating luminescence in thepresence of ATP” as used herein refers to a single reagent orcombination of components that, in the presence of ATP, will generateluminescence. The amount of luminescence may be reliably related to theamount of ATP present. An example of a reagent suitable for use in thepresent invention is the combination of luciferin and luciferase asdescribed by Crouch et al. (J. Immunol. Meth. 160, 81-88 (2000)) andBradbury et al. (J. Immunol. Meth. 240, 79-92 (2000) incorporated hereinby reference in their entireties.

[0058] The term “toxicity” as used herein refers to the ability of acompound or a combination of compounds to negatively modulate theproliferation of a population of hematopoietic stem or progenitor cells.It will be understood that the toxicity of a compound or compounds maybe effective against one hematopoietic cell lineage and not againstanother, and may further include the ability of a compound to modulatethe differentiation of a hematopoietic stem or progenitor cell.

[0059] The term “differentially distinguishable” as used herein refersto hematopoietic stem and progenitor cells, or any other animal cell,the proliferation status of which may be usefully determined by theassay methods of the present invention and which can be characterizedinto subpopulations based on, for example, different complements of cellsurface markers.

[0060] Following longstanding law convention, the terms “a” and “an” asused herein, including the claims, are understood to mean “one” or“more”.

[0061] Abbreviations

[0062] Abbreviations used in the present specification include thefollowing: IL, interleukin; PBMC, peripheral blood mononuclear cells;PBS, phosphate-buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mMKCl, pH 7.4); FBS, fetal bovine serum; BSA, bovine serum albumen

[0063] Reference now will be made in detail to the aspects andembodiments of the invention. Each example is provided by way ofexplanation of the invention, and not a limitation of the invention. Infact, it will be apparent to those skilled in the art that variousmodifications, combination, additions, deletions and variations can bemade in the present invention without departing from the scope or spiritof the invention. For instance, features illustrated or described aspart of one embodiment can be used in another embodiment to yield astill further embodiment. It is intended that the present inventioncovers such modifications, combinations, additions, deletions andvariations as come within the scope of the appended claims and theirequivalents.

[0064] The high-throughput assay methods of the present inventioncomprise the detection and enumeration of a population of hematopoieticcells by measuring the metabolic activity of samples of proliferatingcells as indicated by their ATP content. The ATP content can be measuredby detecting the luminescence generated by a ATP-dependent reactionrequiring, for example, contacting the cells with an ATP-releasing agentand an ATP-monitoring agent. A suitable system for detecting ATP by theemission of luminescence comprises the combination of luciferin andluciferase, although it is contemplated that any method that will emit adetectable signal, the intensity of which may be correlated to theamount of ATP in a cell culture may be within the scope of the presentinvention.

[0065] The high-throughput assay method of the present invention allowsfor the detection of actively proliferating subpopulations ofhematopoietic stem and progenitor cell lineages that have been inducedto undergo proliferation by exposure of the cell population to one ormore cytokines. Most hematopoietic cell lineages can be induced toproliferate by contacting the cell population with at least onecytokine. It is, therefore, contemplated that a cytokine, or combinationof cytokines, may be selected to induce the proliferation of a selectedcell lineage. It is further contemplated to be within the scope of theassay methods of the present invention for a plurality of primitivehematopoietic stem or progenitor cell populations to be contacted with aplurality of cytokines or combinations of cytokines, therebyestablishing populations of different proliferating cell lineages. Thevarious proliferating cell lineages may then be used as target cells andcontacted with one or more test compounds. The cell proliferationmodulating activities, including toxicity, of the compounds orcombinations and/or doses thereof may be compared and contrasted, aswell as how the various cell lineages will react to the test compounds.

[0066] High-throughput assays of hematopoietic stem and progenitor cellproliferation

[0067] Primitive hematopoietic cells can be isolated from suitableanimal or human tissues including, for example, peripheral blood, bonemarrow, or umbilical cord blood. Mononuclear cells, for exampleperipheral blood mononuclear cells (PBMCs) may be further isolated bymethods such as density-gradient centrifugation. It is contemplated tobe within the scope of the present invention for the primitive cellpopulation to be further subdivided into isolated subpopulations ofcells that are characterized by specific cell surface markers. Themethods of the present invention may further include the separation ofcell subpopulations by methods such as high-speed high-speed cellsorting, typically coupled with flow cytometry.

[0068] For example, the channels of a flow-cytometer and high-speed cellsorter could be set at 530 nm, typically used for FITC labeling, 670 nmused for APC labeling, and a UV channel, for Hoechst (Ho) 33342 or DAPIstaining. Fluorescent compensation software such as the System II orExpo 32 (Beckman Coulter) can allow full use of all of these channels.Cell subpopulations can be selected based on the presence or absence ofcell membrane antigen markers, the intracellular pH, and the cell cyclestatus. Exemplary methods for selectively distinguishing subpopulationsof hematopoietic cells are described, for example, in PCT applicationSerial No: 20010012620, incorporated herein in by reference in itsentirety.

[0069] Multiparameter analysis may be conducted on primary normal andleukemic samples or leukemic cell lines. The methods of the presentinvention, however, may be applied or adapted to any non-leukemichematopoietic stem or progenitor cell population that might include asubpopulation of proliferating cells. An antigen indicator conjugated toAPC can be used to selectively detect a normal blood stem cellsubpopulation. Aliquots of cells may be labeled with panels comprisingmore than one biomarker. An example of one such panel incorporatesCD38-FITC, CD34-APC, SNARF and Ho33342. Other examples of possiblepanels can include substituting CD38-FITC with CD117(c-kit)-FITC, withCD91 (Thy-1)-FITC or with AC 133-FITC.

[0070] The procedures of the present invention, therefore, can providetechniques to analyze combinations of cell markers as described above,or those specific for other lympho-hematopoietic lineages todifferentiate the effects of inhibitors on normal different cellsubpopulations. A similar reasoning can be applied to leukemic cellpopulations that also show aberrant flow cytometric profilesdistinguishable from the normal population. A typical example would bechronic myeloid leukemia in chronic phase. However, in the case of ALL,the leukemic cell population can be defined by a high proportion ofCD19⁺ cells. Therefore, CD19 is a biomarker that can be used todifferentiate between leukemic and non-leukemic populations. Theselected cell subpopulations can then be applied to the high-throughputassays of the present invention, as described in Examples 1 and 2 below.

[0071] Cell surface indicators may be contacted with the hematopoieticstem or progenitor cells or leukemic cells thereof and the varioussubpopulations may be selectively separated by techniques such as flowcytometry or by attaching the cell surface indicators directly orindirectly to a separable solid support such as magnetic beads. Thebeads and the attached cells thereon can be isolated by a magneticfield.

[0072] In the methods of the present invention as described, forexample, in Examples 1 and 2 below, target hematopoietic stem and/orprogenitor cells are isolated from animal or human tissues and suspendedat cell concentrations ranging from about 1-5×10² to about 1-2×10⁵/ml.Since typical assay volumes are 100 μl, actual cell concentrations inthe assay test vessels will be diluted to {fraction (1/10)} of theoriginal starting cell concentration. The cells are mixed and suspendedin methyl cellulose containing 0% to about 30% concentration of fetalbovine serum (FBS), 1% detoxified bovine serum albumin (BSA),iron-saturated human transferrin at a final concentration of 1×10⁻¹⁰mol/L, α-thioglycerol at a final concentration of 1×10⁻⁴ mol/L andcytokines/growth factors. The methyl cellulose concentration in theassays of the present invention is between about 0.4% and about 0.7%,with a preferred concentration for most cell populations of about 0.7%.One exemplary medium is Iscove's Modified Dulbecco's Medium (IMDM, LifeTechnologies, Rockville, Md.) although other suitable media capable ofsupporting the growth of hematopoietic cells may also be used. Low fetalbovine serum concentrations of between 0% and 10% can also be used. Whenthe assay methods of the present invention are used under serum-freeconditions, insulin (10 μg/ml) and, where necessary, low densitylipoproteins (40 μg/ml) can replace the FBS.

[0073] A stock cell culture is aliquoted into sample chambers. Whilesample chambers may be the wells of a multi-well tissue culture plate,such as a 48- or 96-well plate, it is also contemplated to be within thescope of the present invention to conduct the assays of the presentinvention in any other suitable reaction vessels including, but notlimited to, individual tubes, wells of plates and the like. Cultureplates with a well surface area of about 35 mm² and a low ring of about2 mm high are especially useful and allow colonies to be counted thatare against the wall of the ring. Preferably the sample chambers are nottissue culture treated. For luminescence assays to be performed,multi-well plates that reduce background light emission or scatter whenthe plates are being enumerated in the plate reader may also be used.While it is desirable to use replicate reactions, it is to be understoodthat a single reaction sample may be used for determining theproliferative status of cells for each data point. However, replicatereactions are to be preferred wherever an increase in accuracy isnecessary. For example, reactions may be replicated once, twice or moretimes, including on a single multi-well plate, although quadruplereactions are preferred.

[0074] The assay methods of the present invention especially contemplatethat the cultures can be incubated in a humidified atmosphere having alow oxygen tension for a period preferably extending to about 10 daysbut also to at least about 14 days. A suitable oxygen concentrationrange is from about 3.5% oxygen to about 7.5% oxygen, most preferablyabout 5.0% oxygen, and further comprising about 5% CO₂ as described byBradley et al. (J. Cell Physiol. 97, 517-522 (1968) and Rich & Kubanek(Exp. Hemat. 52, 579-588 (1982) incorporated herein by reference intheir entireties.

[0075] Regardless of the instrument parameters used, there is a directcorrelation between the cell concentration plated in the wells and themean, or sum, of the relative luminescence units obtained. To avoidlarge standard deviations, an integration time of 1000 ms may be used,although other integration times may usefully be selected.

[0076] The high-throughput assay method of the present invention furtherincludes contacting a hematopoietic stem or progenitor cell populationwith at least one cytokine that can induce the proliferation of the stemor progenitor cell population. It is contemplated that the cytokine of acombination of cytokines may be selected to induce the differentiationand proliferation of selected subpopulations of hematopoietic celllineages. Exemplary cytokines include, but are not limited toerythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor. Additional growth factors may also beincluded to boost the proliferative status of a particular culture ofcells including such factors as insulin-like growth factor, insulin andrecombinant insulin. Examples of cytokines or combinations thereof thatmay be used in the assay methods of the present invention and thespecific targeted stem, progenitor or precursor cell types, and theresulting expanded cell lineages are given in Example 3 and Table 1below.

[0077] High-throughput assay methods for toxicity testing withhematopoietic stem and progenitor cells

[0078] It is further contemplated that a cell lineage that is induced toproliferate by contacting a first primitive hematopoietic stem orprogenitor cell population with a cytokine or combination of cytokinesmay further be contacted with a test compound that may have a cytotoxiceffect or a cell proliferation enhancing effect. The degree ofmodulation of cell proliferation or differentiation may also bedetermined by comparing the proliferation of the cell lineage in thepresence of the test compound, and in its absence from the culture of asecond targeted cell population or plurality of second cell populations.It is within the scope of the assay methods of the present invention fora plurality of test compounds to be compared for their cytotoxic effectson one, or a plurality, of proliferating target cell lineages. To theseends, a plurality of hematopoietic stem or progenitor cell populationsmay, for example, be plated in the wells of a multi-well plate or inindividual chambers, thereby allowing rapid testing of multiple samples.

[0079] It is also contemplated that the high-throughput assays of thepresent invention may be used to determine the ability of a testcompound to increase the proliferation of a population of hematopoieticstem or progenitor cells. Such proliferation enhancing compoundsinclude, for example, cytokines and growth factors.

[0080] It is further contemplated that the assay methods of the presentinvention may be used with a range of concentrations of the testcompound which may be contacted with a plurality of cell populations ofthe same cell lineage, whereupon the IC50 or the IC90 for the testcompound acting against the targeted cell population or a subpopulationthereof may be calculated.

[0081] High-throughput assay methods for screening hematopoietic stemand progenitor cell populations for suitability for transplantation

[0082] The high-throughput assay methods of the present invention arealso suitable for screening a population of hematopoietic stem orprogenitor cells to determine the proliferation status of the cells orsubpopulations thereof wherein the proliferative status will indicatethe suitability of the stem or progenitor cells for transplantation intoa recipient animal or human host. The high-throughput assay of thepresent invention will allow the selection of populations of primitivehematopoietic cell that will likely proliferate and maintain engrafmentwithin the recipient patient.

[0083] By determining the sum or mean of the relative luminescenceunits(RLU) in all replicates of a single sample at a specified timepoint during the incubation procedure, for example at 4, 7, 10 or 14days of incubation, the assay can be used to rapidly and quantitativelydetermine: (a) the proliferative status of a hematopoietic stem orprogenitor cell population or of cells of a specific progenitor anddifferentiation lineage and compare such in parallel assays; (b) ifcells from a particular source exhibit a normal or abnormalproliferative capacity; and (c) whether a compound (e.g. growth factor,cytokine, drug, neutraceutical, environmental agent), will have apositive or negative effect of the proliferative status of the cells ina particular cell population. The assay, even of multiple samples, canbe completed within 30 min, calculated from the time of adding the ATPreleasing agent to the conclusion of the luminescence measurement.

[0084] The high-throughput stem/progenitor cell assay (HT-SPCA) of thepresent invention does not count colonies or differentiate betweencolony types. Rather, the HT-SPCA of the present invention measures theproliferation status of cells within the colonies by determining theamount of ATP being produced by the cells. With colony growth in themethyl cellulose assay system of the present invention, some cells inthe cultures will begin to proliferate and form aggregates or clusters.However, the proliferative status of the cell population may be limiteddue to their late stage of differentiation. Thus, a small colony mayensue within a short incubation period, but cell proliferation willrapidly cease.

[0085] In the assay methods of the present invention, the cultureconditions include α-thioglycerol to maintain molecules in a reducedform, and the cultures are incubated under low oxygen tension of betweenabout 3.5% oxygen and about 7.5% oxygen, both conditions reducing oxygentoxicity. The cell aggregate or colony can be maintained in a stagnantor non-proliferative state for between about 2 and about 3 weeks. Othercells, however, that are developmentally more primitive, for example,stem and progenitor cells, have a greater proliferative capacity andwill begin to form colonies after a certain lag period of time. Thesecells will continue to divide throughout the whole of the incubationperiod. Eventually, the proliferative capacity of the cells within thesecolonies will also decrease and finally cease.

[0086] This ability of the assay method of the present invention todistinguish primitive hematopoietic cells from more mature,differentiated lineages contrasts with the conventional manual assaymethods. In the manual assay, in which colonies are counted under amicroscope, proliferating cells cannot be readily distinguished fromnon-proliferating cells. The size of the colony, however, may indicatethe “primitiveness” of the cell that gave rise to that colony. Thus, thelarger the colony, the greater the possibility of the colony derivingfrom a more primitive cell. In the HT-SPCA method of the presentinvention, the size of the colony of the present invention, however, isirrelevant. Rather, it is the proliferative status of the cells withinthe colonies within the same well that is measured, as documented inExample 3 below.

[0087] One aspect of the present invention, therefore, is ahigh-throughput assay method for rapidly determining the proliferativestatus of a population of primitive hematopoietic cells, the methodcomprising the steps of providing a cell population comprising primitivehematopoietic cells, incubating the cell population in a cell growthmedium comprising a concentration of fetal bovine serum between 0% andabout 30% and a concentration of methyl cellulose between about 0.4% andabout 0.7%, and in an atmosphere having between about 3.5% oxygen about5.5% oxygen, preferably 5.0% oxygen, contacting the cell population witha reagent capable of generating luminescence in the presence of ATP, anddetecting luminescence generated by the reagent contacting the cellpopulation, the level of luminescence indicating the amount of ATP inthe cell population, wherein the amount of ATP indicates theproliferative status of the primitive hematopoietic cells.

[0088] In one embodiment of the method of the present invention, theconcentration of fetal bovine serum is between about 0% and 10%.

[0089] In another embodiment of the method of the present invention, theconcentration of methyl cellulose is about 0.7%.

[0090] In yet another embodiment of the present invention, theconcentration of oxygen in the atmosphere is about 5%.

[0091] Another embodiment of the method of the present invention furthercomprises the step of contacting the primitive hematopoietic cellpopulation with at least one cytokine and optionally may furthercomprise the step of generating a cell population substantially enrichedin hematopoietic stem cells.

[0092] One embodiment of the method of the present invention comprisesthe step of generating a cell population substantially enriched in atleast one hematopoietic progenitor cell lineage.

[0093] In one embodiment of the method of the present invention, theprimitive hematopoietic cells are hematopoietic stem cells.

[0094] In another embodiment of the method of the present invention, theprimitive hematopoietic cells are hematopoietic progenitor cells.

[0095] In yet another embodiment of the method of the present invention,the population of primitive hematopoietic cells comprises hematopoieticstem cells and hematopoietic progenitor cells.

[0096] In still another embodiment of the method of the presentinvention, the primitive hematopoietic cells are primary hematopoieticcells.

[0097] In one embodiment of the method of the present invention, theprimary hematopoietic cells are isolated from animal tissue selectedfrom the group consisting of peripheral blood, bone marrow, umbilicalcord blood, yolk sac, fetal liver and spleen.

[0098] In one embodiment of the method of the present invention, theanimal tissue is obtained from a human.

[0099] In one embodiment of the method of the present invention, theanimal tissue is selected from bone marrow, yolk sac, fetal liver andspleen.

[0100] In various embodiments of the method of the present invention,the animal is a mammal.

[0101] In various embodiments of the method of the present invention,the mammal is selected from the group consisting of cow, sheep, pig,horse, goat, dog, cat, non-human primates, rodents, rabbit and hare.

[0102] In another embodiment of the method of the present invention, theanimal tissue is human tissue further selected from the group consistingof peripheral blood, bone marrow, umbilical cord blood fetal liver andspleen.

[0103] In yet another embodiment of the method of the present invention,the primary hematopoietic stem cells are isolated from peripheral blood.

[0104] Still another embodiment of the method of the present inventionfurther comprises the step of selecting a differentially distinguishablesubpopulation of primitive hematopoietic cells from the population ofprimitive hematopoietic cells, wherein the subpopulation of cells isdefined by cell surface markers thereon.

[0105] In one embodiment of the method of the present invention, thestep of selecting a differentially distinguishable subpopulation ofprimitive hematopoietic cells from the population of primitivehematopoietic cells comprises the steps of contacting the population ofprimitive hematopoietic cells with at least one cell surface markerindicator capable of selectively binding to a cell surface marker of adifferentially distinguishable subpopulation of cells, and selectivelyisolating the at least one subpopulation of cells binding the at leastone indicator.

[0106] In one embodiment of the method of the present invention, thecell surface marker is selected from the group consisting of CD3, CD4,CD8, CD34, CD90 (Thy-1) antigen, CD117, CD38, CD56, CD61, CD41,glycophorin A, HLA-DR, AC133 defining a subset of CD34⁺ cells, CD19, andHLA-DR.

[0107] In one embodiment of the method of the present invention, thecell surface marker is CD34⁺.

[0108] In one embodiment of the method of the present invention, thesubpopulation of differentially distinguishable primitive cells isselectively isolated by magnetic bead separation.

[0109] In another embodiment of the method of the present invention, thesubpopulation of differentially distinguishable primitive cells isselectively isolated by flow cytometry and cell sorting.

[0110] In yet another embodiment of the method of the present invention,the population of primitive hematopoietic cells comprises at least onestem cell lineage selected from the group consisting of colony-formingcell-blast (CFC-blast), high proliferative potential colony forming cell(HPP-CFC) colony-forming unit-granulocyte, erythroid, macrophage,megakaryocyte (CFU-GEMM).

[0111] In the various embodiments of the methods of the presentinvention, the population of primitive hematopoietic cells comprises atleast one hematopoietic progenitor cell lineage selected from the groupconsisting of granulocyte-macrophage colony-forming cell (GM-CFC),megakaryocyte colony-forming cell (CFC-mega), macrophage colony-formingcell (M-CFC), granulocyte colony forming cell (G-CFC), burst-formingunit erythroid (BFU-E), colony-forming unit-erythroid (CFU-E),colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil(CFC-Eo), colony-forming cell-megakaryocyte (CFC-Mega), B cellcolony-forming cell (B-CFC) and T cell colony-forming cell (T-CFC).

[0112] Also, in the various embodiments of the methods of the presentinvention, the reagent capable of generating luminescence in thepresence of ATP comprises luciferin and luciferase.

[0113] Also, in the various embodiments of the methods of the presentinvention, the at least one cytokine is selected from the groupconsisting of erythropoietin, granulocyte-macrophage colony stimulatingfactor, granulocyte colony stimulating factor, macrophage colonystimulating factor, thrombopoietin, stem cell factor, interleukin-1,interleukin-2, interleukin-3, interleukin-6, interleukin-7,interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growthfactor, and insulin.

[0114] In one embodiment of the method of the present invention, the atleast one cytokine is stem cell factor, interleukin-7 and Flt3L, andwherein the at least one cytokine generates a cell populationsubstantially enriched in colony-forming cells blast (CFC-Blast) stemcells.

[0115] In another embodiment of the method of the present invention, theat least one cytokine is macrophage colony stimulating factor,interleukin-1, interleukin-3, interleukin-6 and stem cell factor, andwherein the at least one cytokine generates a cell populationsubstantially enriched in hematopoietic high proliferative potentialcolony-forming cell (HPP-CFC) stem cells.

[0116] In yet another embodiment of the method of the present invention,the at least one cytokine is erythropoietin, granulocyte-macrophagecolony stimulating factor, granulocyte colony stimulating factor, stemcell factor, interleukin-3, interleukin-6, and optionally Flt3L, andwherein the at least one cytokine generates a cell populationsubstantially enriched in hematopoietic colony-forming cell erythroid,macrophage, megakaryocyte (CFC-GEMM) stem cells.

[0117] In still another embodiment of the method of the presentinvention, the at least one cytokine is selected from the groupconsisting of erythropoietin, erythropoietin and interleukin-3,erythropoietin and stem cell factor and erythropoietin, stem cell factorand interleukin-3, and wherein the at least one cytokine generates acell population substantially enriched in the hematopoietic burstforming unit-erythroid (BFU-E) progenitor cells.

[0118] In still yet embodiment of the method of the present invention,the at least one cytokine is further selected fromgranulocyte-macrophage colony stimulating factor, granulocyte-macrophagecolony stimulating factor and interleukin-3, and granulocyte-macrophagecolony stimulating factor, interleukin-3 and stem cell factor, andwherein the at least one cytokine generates a cell populationsubstantially enriched in hematopoietic granulocyte-macrophagecolony-forming cell (GM-CFC) progenitor cells.

[0119] In another embodiment of the method of the present invention, theat least one cytokine is further selected from the groups consisting ofthrombopoietin, and thrombopoietin, interleukin-3 and interleukin-6, andwherein the at least one cytokine generates a cell populationsubstantially enriched in the hematopoietic megakaryocyte colony-formingcell (CFC-Mega) progenitor cells.

[0120] In yet another embodiment of the method of the present invention,the at least one cytokine is further selected from interleukin-2, andinterleukin-7, Flt3L and interleukin-15, and wherein the at least onecytokine generates a cell population substantially enriched in thehematopoietic T cell colony forming cell (T-CFC) progenitor cells.

[0121] In still another embodiment of the method of the presentinvention, the at least one cytokine is selected from the groupconsisting of interleukin-7, and interleukin-7 and Flt3L, and whereinthe at least one cytokine generates a cell population substantiallyenriched in the hematopoietic B cell colony-forming cell (B-CFC)progenitor cells.

[0122] In still yet another embodiment of the method of the presentinvention, the at least one cytokine is erythropoietin and wherein theat least one cytokine generates a cell population substantially enrichedin the hematopoietic colony-forming unit-erythroid (CFU-E) progenitorcells.

[0123] In another embodiment of the method of the present invention, theat least one cytokine is selected from the group consisting ofgranulocyte-colony stimulating factor and granulocyte-macrophage colonystimulating factor, and wherein the at least one cytokine generates acell population substantially enriched in the hematopoietic granulocytecolony-forming cell (G-CFC) progenitor cells.

[0124] In yet another embodiment of the method of the present invention,the at least one cytokine is selected from the group consisting ofinterleukin-3, and interleukin-3 and stem cell factor, and wherein theat least one cytokine generates a cell population substantially enrichedin the hematopoietic colony-forming cell-Basophil (CFC-Bas) progenitorcells.

[0125] In still another embodiment of the method of the presentinvention, the at least one cytokine granulocyte-macrophage colonystimulating factor, interleukin-3 and interleukin-5, and wherein the atleast one cytokine generates a cell population substantially enriched inthe hematopoietic colony-forming cell-eosinophil (CFC-Eo) progenitorcells.

[0126] In still yet another embodiment of the method of the presentinvention, the at least one cytokine is selected from the groupconsisting of macrophage colony stimulating factor, macrophage colonystimulating factor and granulocyte-macrophage colony stimulating factorand interleukin-7, and granulocyte-macrophage colony stimulating factor,and wherein the at least one cytokine generates a cell populationsubstantially enriched in the hematopoietic macrophage colony-formingcell (M-CFC) progenitor cells.

[0127] One embodiment of the method of the present invention furthercomprises the step of identifying a population of primitivehematopoietic cells having a proliferative status suitable fortransplantation into a recipient patient.

[0128] Another embodiment of the method of the present invention furthercomprises the steps of providing a population of primitive hematopoieticcells comprising a target cell population, contacting the target cellpopulation with a test compound, and determining the ability of the testcompound to modulate the proliferative status of the target cellpopulation.

[0129] In one embodiment of the method of the present invention, thepopulation of primitive hematopoietic cells comprises a plurality oftarget cell populations, and the method further comprises the steps ofcontacting the plurality of target cell populations with at least onetest compound, determining the ability of the at least one test compoundto alter the proliferation of the target cell population by comparingthe proliferative status of the plurality of target cell populationswith the proliferative status of a target population of primitivehematopoietic cells not in contact with the test compound, andidentifying the at least one test compound modulating the proliferativestatus of a target cell population.

[0130] Another aspect of the present invention, therefore, is ahigh-throughput assay method for rapidly identifying a population ofprimitive hematopoietic cells having a proliferative status suitable fortransplantation into a patient, comprising the steps providing a cellpopulation comprising primitive hematopoietic cells, incubating the cellpopulation in cell a growth medium comprising between 0% and 30% fetalbovine serum and a concentration of methyl cellulose between about 0.4%and about 0.7%, and in an atmosphere having between about 3.5% and about7.5% oxygen, contacting the primitive hematopoietic cell population withat least one cytokine selected from the group consisting oferythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin,contacting the cell population with a reagent capable of generatingluminescence in the presence of ATP, and detecting luminescencegenerated by the reagent contacting the at least two cell populations,the level of luminescence indicating the proliferative status of theprimitive hematopoietic cells, and wherein the proliferative status ofthe primitive hematopoietic cells indicates the suitability of the cellpopulation for transplantation into a recipient patient.

[0131] In one embodiment of this aspect of the method of the presentinvention, contacting the population of primitive hematopoietic cellswith at least one cytokines generates a cell population substantiallyenriched in a hematopoietic stem cell lineage.

[0132] In one embodiment of the method of the present invention, thehematopoietic stem cell lineage is selected from the group consisting ofcolony-forming cell-blast (CFC-blast), high proliferative potentialcolony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).

[0133] In another embodiment of this aspect of the method of the presentinvention, contacting the population of primitive hematopoietic cellswith at least one cytokine generates a cell population substantiallyenriched in at least one hematopoietic progenitor cell lineage.

[0134] In the various embodiments of this aspect of the method of thepresent invention, the population of primitive hematopoietic cellscomprises at least one hematopoietic progenitor cell lineage selectedfrom the group consisting of granulocyte-macrophage colony-forming cell(GM-CFC), megakaryocyte colony-forming cell (CFC-mega), macrophagecolony-forming cell (M-CFC), granulocyte colony forming cell (G-CFC),burst-forming unit erythroid (BFU-E), colony-forming unit-erythroid(CFU-E), colony-forming cell-basophil (CFC-Bas), colony-formingcell-eosinophil (CFC-Eo), colony-forming cell-megakaryocyte (CFC-Mega),B cell colony-forming cell (B-CFC) and T cell colony-forming cell(T-CFC).

[0135] Yet another aspect of the present invention is a high-throughputassay method for rapidly identifying a compound capable of modulatingthe proliferative status of a population of primitive hematopoieticcells, comprising the steps of providing a first target cell populationcomprising primitive hematopoietic cells, incubating the cell populationin cell a growth medium comprising between 0% and 30% fetal bovine serumand a concentration of methyl cellulose between about 0.4% and about0.7%, and in an atmosphere having between about 3.5% and about 7.5%oxygen, providing a plurality of second target cell populationscomprising primitive hematopoietic cells, contacting the first andsecond target primitive hematopoietic cell populations with at least onecytokine selected from the group consisting of erythropoietin,granulocyte-macrophage colony stimulating factor, granulocyte colonystimulating factor, macrophage colony stimulating factor,thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin,contacting the first and second target cell populations with at leastone test compound, contacting the first and second target cellpopulations with a reagent capable of generating luminescence in thepresence of ATP, detecting luminescence generated by the reagentcontacting the first and second target cell populations, the level ofluminescence indicating the proliferative status of the primitivehematopoietic cells, and comparing the proliferative status of theplurality of the second target cell populations with the proliferativestatus of the first target population of primitive hematopoietic cellsnot in contact with the test compound, thereby identifying a testcompound capable of modulating the proliferative status of a target cellpopulation.

[0136] In the various embodiments of this aspect of the method of thepresent invention, contacting the first and second target populations ofprimitive hematopoietic cells with at least one cytokine generates cellpopulations substantially enriched in hematopoietic stem cells.

[0137] In other embodiments of the methods of the present invention, thehematopoietic stem cells are selected from the group consisting ofcolony-forming cell-blast (CFC-blast), high proliferative potentialcolony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).

[0138] Also, in the various embodiments of this aspect of the method ofthe present invention, contacting the first and second targetpopulations of primitive hematopoietic cells with at least one cytokinegenerates cell populations substantially enriched in at least onehematopoietic progenitor cell lineage.

[0139] In the various embodiments of this aspect of the method of thepresent invention, the at least one hematopoietic progenitor celllineage selected from the group consisting of granulocyte-macrophagecolony-forming cell (GM-CFC), megakaryocyte colony-forming cell(CFC-mega), macrophage colony-forming cell (M-CFC), granulocyte colonyforming cell (G-CFC), burst-forming unit erythroid (BFU-E),colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil(CFC-Bas), colony-forming cell-eosinophil (CFC-Eo), colony-formingcell-megakaryocyte (CFC-Mega), B cell colony-forming cell (B-CFC) and Tcell colony-forming cell (T-CFC).

[0140] One embodiment of the method of the present invention furthercomprises the steps of contacting a target cell population with at leasttwo concentrations of a test compound, and calculating the IC50 of thetest compound.

[0141] Another embodiment of the method of the present invention furthercomprises the steps of contacting a target cell population with at leasttwo concentrations of a test compound and calculating the IC90 of thetest compound.

[0142] The present invention is further illustrated by the followingexamples, which are provided by way of illustration and should not beconstrued as limiting. The contents of all references, published patentsand patents cited throughout the present application are herebyincorporated by reference in their entirety.

EXAMPLE 1 Preparation of Incubated Hematopoietic Stem or ProgenitorCells

[0143] Isolation of mononuclear cells

[0144] Mononuclear cells (MNC) were prepared from human peripheralblood, bone marrow or umbilical cord blood by density gradientcentrifugation on Ficoll-Paque Plus by diluting the cell suspension 1:1with sterile PBS and transferring up to 30 ml to a 15-20 ml cushion ofFicoll. Centrifugation was at 400 g for 20 mins at room temperature. Thesupernatant was discarded and the cells were resuspended in 50 ml ofsterile PBS and re-centrifuged at 200 g for 10 mins at room temperature.Thereafter, the supernatant was discarded and the cells were resuspendedin IMDM to ensure a single cell suspension. A cell count was determined.If not used immediately, the cells were placed on ice or at 4° C.

[0145] Cells from peripheral blood mononucleocytes (MNCs) were preparedat a final concentration of 2×10⁶ cells/ml. MNCs from bone marrow andumbilical cord blood were prepared at final concentrations of 0.5-1×10⁶and 0.5-1×10⁵ respectively.

[0146] Bulk reagent solutions

[0147] The following components were mixed in sterile tubes, usually inthe following order, so that the final total volume of the culturemixture was 600 μl or multiples thereof. The volume prepared depended onthe number of replicate assays required. A “master mix” of 600 μl wassufficient for 4-5 replicates of 100 μl each. If volumes of the mastermix greater than 600 μl were required, medium was added first followedby methyl cellulose. This mixture was then mixed on a vortex mixer. Iflarge quantities of methyl cellulose were added first, it becamedifficult to mix the components adequately.

[0148] (a) Serum-containing cultures

[0149] (i) Methyl cellulose (stock at 2.6% v/v),. 160 μl, (finalconcentration, 0.7% v/v)

[0150] (ii) Fetal bovine serum (FBS), 180 μl

[0151] (iii) α: thioglycerol, 6 μl at a final concentration of 1×10⁻⁴M

[0152] (iv) Human or bovine iron-saturated transferrin, 6 μl, finalconcentration of 1×10⁻¹⁰ g/ml

[0153] (v) Growth factors, individually or in combination, were selectedfrom the following: erythropoietin, 1-3 U/ml; granulocyte-macrophagecolony stimulating actor, 10-20 ng/ml; granulocyte colony stimulatingfactor, 10-20 ng/ml; macrophage colony stimulating factor, 10-20 ng/ml;thrombopoietin, 50 ng/ml; stem cell factor, 50 ng/ml; interleukin-1,10-20 ng/ml; interleukin-2, 2-10 ng/ml; interleukin-3, 20 ng/ml;interleukin-6, 20 ng/ml; interleukin-7, 10 ng/ml. The volume addeddepended upon the concentration of the cytokine/growth factor stocksolution, but typically were not greater than 6-10 μl. All growthfactors were diluted in IMDM containing either 5% FBS or 1% BSA

[0154] (vi) Cells diluted in IMDM to the required final concentration asdescribed above and added at 60μl.

[0155] (vii) IMDM added to give a final stock solution volume of 600 μl(or multiples thereof).

[0156] (b) Serum-free cultures.

[0157] For serum-free conditions, the fetal bovine serum of (ii) abovewas replaced by a mixture of bovine serum albumen, transferrin andinsulin added as a preformed mixture (BIT 9500, Stem Cells Technologies,Vancouver).

[0158] Once the basic components were added, including the growthfactors that were required for the specific cell type to be analyzed,the contents were vortexed to yield a homogenous mixture. The reactionmixes were left to stand for a few minutes before dispensing to thewells of a 96-well plate or other arrays of receptacles. When cultureswere incubated for more than 7 days, the outer wells of a 96-well platewere filled with 100 μl of sterile water to ensure that the cultures didnot desiccate, even when a humidified tissue culture incubator was used.

[0159] Using a 1 ml syringe with an 18 gauge 1.5″ needle, or a repeaterpipette with a syringe capable of dispensing aliquots of 100 μl,serum-containing, or a serum-free culture prepared as described abovewas withdrawn slowly and dispensed into each of the replicate wells of a96-well plate while ensuring that little or none of the master mixtouched the sides of the wells.

[0160] Sample incubation

[0161] Once the samples had been dispensed into the wells, the 96-wellplate was placed in a fully humidified tissue culture incubator at 37°C. The cells were incubated in a low oxygen tension atmosphere of 5% CO₂and 5% oxygen (obtained by replacing the oxygen in the incubator withnitrogen gas). The incubation period depended on the cell population tobe tested.

EXAMPLE 2 Measurement of the ATP Content of Incubated Hematopoietic Stemor Progenitor Cells

[0162] After the incubation time has elapsed, the reagents from theViaLight HS™ kits (Lumitech) were prepared for use. If necessary, thenumber of cell clusters (aggregates) or colonies that had developed inthe wells of the incubated 96-well plates could be counted under aninverted microscope to ensure that a correlation between the sum, ormean, of the ratio of clusters/colonies to the relative luminescenceunits (RLU) was obtained (see below).

[0163] All reagents were allowed to attain room temperature before use.The ATP monitoring reagent was reconstituted as described by themanufacturers by adding 10 ml of the supplied buffer to the lyophilizedreagent and waiting 15 mins. Alternatively, 1 ml of the buffer was usedto reconstitute the reagent and the latter was then aliquoted into 1.5ml microtubes and frozen while protected from light. Aliquots were thenthawed and diluted to 1 ml final volumes using the supplied buffer asneeded. The ATP monitoring reagent was protected from light at alltimes.

[0164] The required quantity of ATP releasing reagent was transferredinto the reagent trough and 100 μl aliquots were transferred, using amulti-tip pipette, to each row or column of wells of the 96-well platedpreviously incubated as described in Example 1 above. After dispensingthe reagent to one row or column, the contents of the wells were mixedat least 4-5 times with the pipette, so that the reagents mixed wellwith the methyl cellulose master mixes. Addition of the reagents dilutedthe methyl cellulose and mixing ensured that the cells came into contactwith the ATP releasing reagent. This step had to be performed in asimilar manner for all wells.

[0165] Once the ATP releasing reagent had been dispensed into all of thewells containing incubated cultures, and mixed therein, the plates weretypically incubated in the dark for 5 min, although the incubation couldproceed for up to 30 min without loss of sensitivity.

[0166] The required amount of ATP monitoring reagent was transferred toa new, clean trough and 20 μl of the reagent pipetted into each of thewells while ensuring that the contents of each well was mixedthoroughly. The plates were immediately transferred to a plate readerand the luminescence measured using an integration time of 1000 ms.

[0167] Measurement was given as relative luminescence units (RLU). Inaddition, since no two makes of luminescence readers are the same, thegain required to obtain sufficient RLU by each machine had to beempirically determined, but could also be standardized between machinesas follows:

[0168] If space allowed on the same plate as the wells containing theincubated cell cultures, 100 μl of a 10⁻⁶ M standard ATP diluted in IMDMfrom a 10⁻⁵ M ATP stock solution was added to each of 4-6 wells. 100 μlof ATP releasing agent was then added and incubated for the same periodas had been used for the cell culture wells. 20 μl of ATP monitoringreagent was then added and the plate transferred to the plate reader andthe luminescence measured at an integration time of 1000 ms. The gainwas adjusted such that the luminescence from the ATP standard wasapproximately 20,000 RLU. By adjusting the gain of the machine to obtainthis number of RLU, and then reading the remaining wells of the plate(containing the incubated cells), no overflow values occurred, therebyobviating the need for a second or multiple reading. In all cases, theluminescence was measured in the shortest possible time period possible,because the luminescence decreased rapidly with time.

[0169] The correlation between the initial plated cell concentration(0.25, 0.5, 0.75, 1, 1.5 2×10⁵/well) and the mean (FIGS. 1A, 2A, 3A and4A respectively) or sum (FIGS. 1B, 2B, 3B, and 4B respectively) ofrelative luminescence units (RLU) measured at 4 days (FIGS. 1A and 1B),7 days (FIGS. 2A and 2B), 10 days (FIGS. 3A and 3B) and 14 days (FIGS.4A and 4B) after culture initiation, as a function of the integrationtime and/or gain of the plate reader. In FIGS. 1A-4B the value 2000represents an integration time of 2000 ms. “Max” represents the maximumintegration time. The values 200, 215, 225 or 250 represent the gainsthat were used with the respective integration times.

EXAMPLE 3 Hematopoietic Stem and Progenitor Cell Lines and TheirAssociated Cytokine Effectors

[0170] Hematopoietic stem and progenotor cells are induced todifferentiate into hematopoietic cell subpopulations by exposure to oneor more growth factors/cytokines, as shown in Table 1 below. TABLE 1Development Population Stimulatory Growth stage Lineage Population nameabbreviation factors and cytokines Stem cell None Long-term culture-LTC-IC Stimulated by (Most initiating cells microenvironmental primitivein cells vitro stem cell) Stem cell None Colony-forming cell- CFC-BlastFlt3L, SCF and IL-7 (Very Blast primitive in vitro stem cell) Stem cellNone High proliferative HPP-CFC IL-1, IL-3, IL-6, SCF, (Primitive inpotential colony- M-CSF vitro stem forming cell cell) Stem cell NoneColony-forming cell CFC-GEMM IL-3, IL-6, GM-CFC, G- (Most maturegranulocyte, CSF, EPO, and SCF in vitro stem erythroid and/or Flt3Lcell) macrophage, megakaryocyte Progenitor Erythroid Burst-forming unit-BFU-E EPO Erythroid IL-3 and EPO SCF and EPO IL-3, SCF and EPOProgenitor Granulocyte- Granulocyte- GM-CFC GM-CSF Macrophage macrophagecolony- IL-3 and GM-CSF forming unit IL-3, SCF and GM-CSF ProgenitorMegakaryocy Megakaryocyte CFC-Mega TPO te colony-forming cell IL-3, IL-6and TPO Progenitor T T cell colony-forming T-CFC IL-2 lymphocyte cellIL-7, Flt3L and IL-15 Progenitor B B cell colony- B-CFC IL-7 lymphocyteforming cell IL-7 and Flt3L Precursor Erythroid Colony-forming cell-CFU-E EPO erythroid Precursor Myeloid- Granulocyte colony- G-CFC G-CSFNeutrohil forming cell GM-CSF, high concentrations Precursor Myeloid-Colony-forming cell- CFC-Bas IL-3 Basophil basophil IL-3 and SCFPrecursor Myeloid- Colony-forming CFC-Eo GM-CSF and IL-5 and Eosinophilcells-eosinophil IL-3 Precursor Macrophage Macrophage colony- M-CFCM-CSF forming cell M-CSF, GM-CSF, IL-3 GM-CSF, low concentrations

EXAMPLE 4 Proliferation of Hematopoietic Stem and Progenitor CellsMeasured by Colony Counting and ATP Determination

[0171] When cell proliferation was measured as a function of time inculture, some aggregates or colonies contained cells that wereproliferating, while others were not, as shown in FIGS. 5A-5C. Wells,therefore, could contain few colonies, but still exhibit high cellproliferation. The results shown in FIGS. 5A-5C show that the number ofcell clusters counted per well does not correlate with the cellproliferation as detected using the luminescence of the presentinvention.

[0172] In contrast, in those wells in which minimal or no clusterformation was detected, luminescence could be detected. In some wells,the luminescence was significantly greater than expected from the numberof cell clusters counted, indicating that cell proliferation wasoccurring and that the proliferating cells, were primitive because oftheir increased proliferative capacity. On day 10, most wells containedcells that were proliferating. By day 14, this proliferative capacitywas only seen in some wells, indicating that proliferation has ceased(RLU lower than the cell cluster count) or is declining. Those wellsexhibiting a significantly greater RLU than determined by manual cellcluster counting showed that cells were present that were capable ofextensive proliferation and were probably stem cells.

[0173] Little or no correlation existed between the number of individualcolonies and the luminescence, as shown in FIGS. 6A-6C. However, underthe typical incubation and culture conditions of the assay of thepresent invention, a well containing a few colonies, but with highindicated cell proliferation, showed that the cells in the culture havehigh proliferative capacity. In other words, the cells were primitive innature.

[0174] The number of colonies in a well did not generally correlate withthe RLU from that well, as shown in FIGS. 6A-6C. In contrast, however,in the high-throughput assay method of the present invention, theluminescence measurements were made over the whole area of each andevery replicate well, and not from the individual cell aggregates orcolonies within these areas. The sum of the luminescent values ofaggregates or colonies, or the mean of the aggregates or colonies fromall replicate wells, can be predicted to correlate with the sum or meanof the luminescence emitted from the replicate wells, as shown in FIGS.7A-7C.

[0175] There was a direct correlation between the sum, or mean, of allthe cell aggregates or colonies from all replicate wells and the sum ormean of the RLU from all replicate wells. Furthermore, this relationshipwas cell dose dependent, as illustrated in FIGS. 8A-8C.

[0176] These results, as opposed to those in FIGS. 6A-6C indicate thatusing the sum or the mean of the replicates from particular sample, inthis case, replicates at different cell concentrations, a directcorrelation exists between the 3 parameters. If measurement ofluminescence is depicted as the sum or the mean of the replicates, therewas also be a direct correlation with the sum and/or mean of the cellclusters, as shown in FIGS. 8A-8D.

[0177] To validate the 96-well plate assay, experiments using both assaysystems were performed in parallel. A direct correlation exists betweenthe 4-well and the 96-well plate assay, as shown in FIGS. 9A-9C andindicates that the results obtained from the latter have been validated.

EXAMPLE 5 Use of High-Throughput Stem/Progenitor Cell Assays toDetermine the Ability of a Test Compound to Modulate the Proliferationof Hematopoietic Stem and Progenitor Cells

[0178] The HT-SPCA of the present invention is used to test doseresponses for a variety of compounds that can interact withhematopoietic stem and progenitor cells. The agents either stimulate orinhibit and/or kill hematopoietic cells. Increasing doses of an agentcan stimulate cells, but then be inhibitory by being toxic and causingnecrosis. Other agents can be toxic at high doses, but induce apoptosisat lower concentrations.

[0179] Agents with known action on hematopoietic stem and progenitorcells include, 5-fluorouracil (5-FU), hydroxyurea, cytosine arabinoside(ara-C). busulphan, 3′azido-3′deoxythymide (AZT), cycloheximide,actinomycin D, etoposide, BCNU, doxorubicin, cisplatin (lowhemotoxicity) and carboplatin. Growth factors known to inhibit theproliferation of stem and progenitor cells such as interferon-γ (IFNγ),tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGFβ)are also tested. Neutraceuticals include, for example, theanti-inflammatory phytochemicals, black and green tea polyphenols,resveritrol, limonene and curcumin.

[0180] For these and other agents to be tested, mononuclear cellsderived from peripheral blood, bone marrow and cord blood are used.CD34+ cells derived from these tissues can also be used. HT-SPCA assaysfor CFU-GEMM, GM-CFC, BFU-E, CFC-Mega and CFU-E, CFC-blast, HPP-CFC,M-CFC and G-CFC induced to proliferate and differentiate by contactingthe cell populations with the appropriate cytokine of combinations ofcytokines as given in Example 3, Table 1, above, are also performed.

[0181] HT-SPCA can be used to detect and predict hemotoxicity againsthematopoietic stem and progenitor cell populations. To validate theinhibition/hemotoxicity of the agents, both manual CFA and the HT-SPCAare performed in parallel. One of the end points is to determine theIC50 and IC90 for the drug.

What is claimed is:
 1. A high-throughput assay method for rapidlydetermining the proliferative status of a population of primitivehematopoietic cells, the method comprising the steps of: (a) providing acell population comprising primitive hematopoietic cells; (b) incubatingthe cell population in a cell growth medium comprising fetal bovineserum having a concentration of between 0% and 30% and methyl cellulosehaving a concentration of between about 0.4% and about 0.7%, and in anatmosphere having between about 3.5% oxygen and 7.5% oxygen; (c)contacting the cell population with a reagent capable of generatingluminescence in the presence of ATP; and (d) detecting luminescencegenerated by the reagent contacting the cell population, the level ofluminescence indicating the amount of ATP in the cell population,wherein the amount of ATP indicates the proliferative status of theprimitive hematopoietic cells.
 2. The method of claim 1, wherein theconcentration of fetal bovine serum is between about 0% and 10%.
 3. Themethod of claim 1, wherein the concentration of methyl cellulose isabout 0.7%.
 4. The method of claim 1, wherein the concentration ofoxygen in the atmosphere is about 5%.
 5. The method of claim 1, furthercomprising the step of contacting the primitive hematopoietic cellpopulation with at least one cytokine.
 6. The method of claim 5, furthercomprising the step of generating a cell population substantiallyenriched in hematopoietic stem cells.
 7. The method of claim 5, furthercomprising the step of generating a cell population substantiallyenriched in at least one hematopoietic progenitor cell lineage.
 8. Themethod of claim 1, wherein the primitive hematopoietic cells arehematopoietic stem cells.
 9. The method of claim 1, wherein theprimitive hematopoietic cells are hematopoietic progenitor cells. 10.The method of claim 1, wherein the population of primitive hematopoieticcells comprises hematopoietic stem cells and hematopoietic progenitorcells.
 11. The method of claim 1, wherein the primitive hematopoieticcells are primary hematopoietic cells.
 12. The method of claim 11,wherein the primary hematopoietic cells are isolated from an animaltissue selected from the group consisting of peripheral blood, bonemarrow, umbilical cord blood, yolk sac, fetal liver and spleen.
 13. Themethod of claim 12, wherein the animal tissue is obtained from a human.14. The method of claim 12, wherein the animal tissue is obtained from amammal.
 15. The method of claim 14, wherein the mammal is selected fromthe group consisting of cow, sheep, pig, horse, goat, dog, cat,non-human primates, rodents, rabbit and hare.
 16. The method of claim14, wherein the animal tissue is selected from bone marrow, yolk sac,fetal liver and spleen.
 17. The method of claim 13, wherein the animaltissue is human tissue further selected from the group consisting ofperipheral blood, bone marrow, umbilical cord blood, fetal liver andspleen.
 18. The method of claim 11, wherein the primary hematopoieticstem cells are isolated from peripheral blood.
 19. The method of claim1, further comprising the step of selecting a differentiallydistinguishable subpopulation of primitive hematopoietic cells from thepopulation of primitive hematopoietic cells, wherein the subpopulationof cells is defined by cell surface markers thereon.
 20. The method ofclaim 19, wherein the step of selecting a subpopulation of primitivehematopoietic cells comprises the steps of: (a) contacting thepopulation of primitive hematopoietic cells with at least one cellsurface marker indicator capable of selectively binding to a cellsurface marker of a differentially distinguishable subpopulation ofcells; and (b) selectively isolating the at least one subpopulation ofcells binding the at least one indicator.
 21. The method of claim 19,wherein the cell surface marker is selected from the group consisting ofCD3, CD4, CD8, CD34, CD90 (Thy-1) antigen, CD117, CD38, CD56, CD61,CD41, glycophorin A and HLA-DR, AC133 defining a subset of CD34+ cells,CD 19, and HLA-DR.
 22. The method of claim 19, wherein the cell surfacemarker is CD34⁺.
 23. The method of claim 20, wherein the subpopulationof differentially distinguishable primitive cells is selectivelyisolated by magnetic bead separation.
 24. The method of claim 20,wherein the subpopulation of differentially distinguishable primitivecells is selectively isolated by flow cytometry and cell sorting. 25.The method of claim 1, wherein the population of primitive hematopoieticcells comprises at least one stem cell lineage selected from the groupconsisting of colony-forming cell-blast (CFC-blast), high proliferativepotential colony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).
 26. The method of claim1, wherein the population of primitive hematopoietic cells comprises atleast one hematopoietic progenitor cell lineage selected from the groupconsisting of granulocyte-macrophage colony-forming cell (GM-CFC),megakaryocyte colony-forming cell (CFC-mega), macrophage colony-formingcell (M-CFC), granulocyte colony forming cell (G-CFC), burst-formingunit erythroid (BFU-E), colony-forming unit-erythroid (CFU-E),colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil(CFC-Eo), colony-forming cell-megakaryocyte (CFC-Mega), B cellcolony-forming cell (B-CFC) and T cell colony-forming cell (T-CFC). 27.The method of claim 1, wherein the reagent capable of generatingluminescence in the presence of ATP comprises luciferin and luciferase.28. The method of claim 5, wherein the at least one cytokine is selectedfrom the group consisting of erythropoietin, granulocyte-macrophagecolony stimulating factor, granulocyte colony stimulating factor,macrophage colony stimulating factor, thrombopoietin, stem cell factor,interleukin-1, interleukin-2, interleukin-3, interleukin-6,interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor,insulin-like growth factor, and insulin.
 29. The method of claim 5,wherein the at least one cytokine is stem cell factor, interleukin-7 andFlt3L, and wherein the at least one cytokine generates a cell populationsubstantially enriched in colony-forming cells blast (CFC-Blast) stemcells.
 30. The method of claim 5, wherein the at least one cytokine ismacrophage colony stimulating factor, interleukin-1, interleukin-3,interleukin-6 and stem cell factor, and wherein the at least onecytokine generates a cell population substantially enriched inhematopoietic high proliferative potential colony-forming cell (HPP-CFC)stem cells.
 31. The method of claim 5, wherein the at least one cytokineis erythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, stem cell factor, interleukin-3,interleukin-6, and optionally Flt3L, and wherein the at least onecytokine generates a cell population substantially enriched inhematopoietic colony-forming cell erythroid, macrophage, megakaryocyte(CFC-GEMM) stem cells.
 32. The method of claim 5, wherein the at leastone cytokine is selected from the group consisting of erythropoietin,erythropoietin and interleukin-3, erythropoietin and stem cell factorand erythropoietin, stem cell factor and interleukin-3, and wherein theat least one cytokine generates a cell population substantially enrichedin the hematopoietic burst forming unit-erythroid (BFU-E) progenitorcells.
 33. The method of claim 5, wherein the at least one cytokine isfurther selected from granulocyte-macrophage colony stimulating factor,granulocyte-macrophage colony stimulating factor and interleukin-3, andgranulocyte-macrophage colony stimulating factor, interleukin-3 and stemcell factor, and wherein the at least one cytokine generates a cellpopulation substantially enriched in hematopoieticgranulocyte-macrophage colony-forming cell (GM-CFC) progenitor cells.34. The method of claim 5, wherein the at least one cytokine is furtherselected from the groups consisting of thrombopoietin, andthrombopoietin, interleukin-3 and interleukin-6, and wherein the atleast one cytokine generates a cell population substantially enriched inthe hematopoietic megakaryocyte colony-forming cell (CFC-Mega)progenitor cells.
 35. The method of claim 5, wherein the at least onecytokine is further selected from interleukin-2, and interleukin-7,Flt3L and interleukin-15, and wherein the at least one cytokinegenerates a cell population substantially enriched in the hematopoieticT cell colony forming cell (T-CFC) progenitor cells.
 36. The method ofclaim 5, wherein the at least one cytokine is selected from the groupconsisting of interleukin-7, and interleukin-7 and Flt3L, and whereinthe at least one cytokine generates a cell population substantiallyenriched in the hematopoietic B cell colony-forming cell (B-CFC)progenitor cells.
 37. The method of claim 5, wherein the at least onecytokine is erythropoietin and wherein the at least one cytokinegenerates a cell population substantially enriched in the hematopoieticcolony-forming unit-erythroid (CFU-E) progenitor cells.
 38. The methodof claim 5, wherein the at least one cytokine is selected from the groupconsisting of granulocyte-colony stimulating factor andgranulocyte-macrophage colony stimulating factor, and wherein the atleast one cytokine generates a cell population substantially enriched inthe hematopoietic granulocyte colony-forming cell (G-CFC) progenitorcells.
 39. The method of claim 5, wherein the at least one cytokine isselected from the group consisting of interleukin-3, and interleukin-3and stem cell factor, and wherein the at least one cytokine generates acell population substantially enriched in the hematopoieticcolony-forming cell-Basophil (CFC-Bas) progenitor cells.
 40. The methodof claim 5, wherein the at least one cytokine granulocyte-macrophagecolony stimulating factor, interleukin-3 and interleukin-5, and whereinthe at least one cytokine generates a cell population substantiallyenriched in the hematopoietic colony-forming cell-eosinophil (CFC-Eo)progenitor cells.
 41. The method of claim 5, wherein the at least onecytokine is selected from the group consisting of macrophage colonystimulating factor, macrophage colony stimulating factor andgranulocyte-macrophage colony stimulating factor and interleukin-7, andgranulocyte-macrophage colony stimulating factor, and wherein the atleast one cytokine generates a cell population substantially enriched inthe hematopoietic macrophage colony-forming cell (M-CFC) progenitorcells.
 42. The method of claim 1, further comprising the step ofidentifying a population of primitive hematopoietic cells having aproliferative status suitable for transplantation into a recipientpatient.
 43. The method of claim 1, wherein a population of primitivehematopoietic cells comprises a target cell population, and furthercomprising the steps of: (i) contacting the target cell population witha test compound; and (ii) determining the ability of the test compoundto modulate the proliferation, and optionally differentiation, of thetarget cell population.
 44. The method of claim 1, wherein thepopulation of primitive hematopoietic cells comprises a plurality oftarget cell populations, and further comprising the steps of: (i)contacting the plurality of target cell populations with at least onetest compound; and (ii) determining the ability of the at least one testcompound to alter the proliferation of the target cell population bycomparing the proliferative status of the plurality of target cellpopulations with the proliferative status of a target population ofprimitive hematopoietic cells not in contact with the test compound; and(iii) identifying the at least one test compound modulating theproliferative status of a target cell population.
 45. A high-throughputassay method for rapidly identifying a population of primitivehematopoietic cells having a proliferative status suitable fortransplantation into a patient, comprising the steps: (a) providing acell population comprising primitive hematopoietic cells; (b) incubatingthe cell population in a cell growth medium comprising a concentrationof fetal bovine serum between 0% and 30% and a concentration of methylcellulose between about 0.4% and about 0.7%, and in an atmosphere havingbetween about 3.5% oxygen and 7.5% oxygen; (c) contacting the primitivehematopoietic cell population with at least one cytokine selected fromthe group consisting of erythropoietin, granulocyte-macrophage colonystimulating factor, granulocyte colony stimulating factor, macrophagecolony stimulating factor, thrombopoietin, stem cell factor,interleukin-1, interleukin-2, interleukin-3, interleukin-6,interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor,insulin-like growth factor, and insulin; (d) contacting the cellpopulation with a reagent capable of generating luminescence in thepresence of ATP; and (e) detecting luminescence generated by the reagentcontacting the at least two cell populations, the level of luminescenceindicating the proliferative status of the primitive hematopoieticcells, and wherein the proliferative status of the primitivehematopoietic cells indicates the suitability of the cell population fortransplantation into a recipient patient.
 46. The method of claim 45,wherein contacting the population of primitive hematopoietic cells withat least one cytokine generates a cell population substantially enrichedin a hematopoietic stem cell lineage.
 47. The method of claim 46,wherein the hematopoietic stem cell lineage is selected from the groupconsisting of colony-forming cell-blast (CFC-blast), high proliferativepotential colony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).
 48. The method of claim45, wherein contacting the population of primitive hematopoietic cellswith at least one cytokine generates a cell population substantiallyenriched in at least one hematopoietic progenitor cell lineage.
 49. Themethod of claim 48, wherein the at least one hematopoietic progenitorcell lineage selected from the group consisting ofgranulocyte-macrophage colony-forming cell (GM-CFC), megakaryocytecolony-forming cell (CFC-mega), macrophage colony-forming cell (M-CFC),granulocyte colony forming cell (G-CFC), burst-forming unit erythroid(BFU-E), colony-forming unit-erythroid (CFU-E), colony-formingcell-basophil (CFC-Bas), colony-forming cell-eosinophil (CFC-Eo),colony-forming cell-megakaryocyte (CFC-Mega), B cell colony-forming cell(B-CFC) and T cell colony-forming cell (T-CFC).
 50. A high-throughputassay method for rapidly identifying a compound capable of modulatingthe proliferative status of a population of primitive hematopoieticcells, comprising the steps: (a) providing a first target cellpopulation comprising primitive hematopoietic cells; (b) incubating thecell population in a cell growth medium comprising a concentration offetal bovine serum between 0% and 30% and a concentration of methylcellulose between about 0.4% and about 0.7%, and in an atmosphere havingbetween about 3.5% oxygen and 7.5% oxygen; (c) providing a plurality ofsecond target cell populations comprising primitive hematopoietic cells;(d) contacting the first and second target primitive hematopoietic cellpopulations with at least one cytokine selected from the groupconsisting of erythropoietin, granulocyte-macrophage colony stimulatingfactor, granulocyte colony stimulating factor, macrophage colonystimulating factor, thrombopoietin, stem cell factor, interleukin-1,interleukin-2, interleukin-3, interleukin-6, interleukin-7,interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growthfactor, and insulin; (e) contacting the first and second target cellpopulations with at least one test compound; and (f) contacting thefirst and second target cell populations with a reagent capable ofgenerating luminescence in the presence of ATP; (g) detectingluminescence generated by the reagent contacting the first and secondtarget cell populations, the level of luminescence indicating theproliferative status of the primitive hematopoietic cells; and (h)comparing the proliferative status of the plurality of the second targetcell populations with the proliferative status of the first targetpopulation of primitive hematopoietic cells not in contact with the testcompound, thereby identifying a test compound capable of modulating theproliferative status of a target cell population.
 51. The method ofclaim 50, wherein contacting the first and second populations ofprimitive hematopoietic cells with at least one cytokine generates cellpopulations substantially enriched in hematopoietic stem cells.
 52. Themethod of claim 51, wherein the hematopoietic stem cells are selectedfrom the group consisting of colony-forming cell-blast (CFC-blast), highproliferative potential colony forming cell (HPP-CFC) colony-formingunit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). 53.The method of claim 50, wherein contacting the first and secondpopulations of primitive hematopoietic cells with at least one cytokinegenerates cell populations substantially enriched in at least onehematopoietic progenitor cell lineage.
 54. The method of claim 53,wherein the at least one hematopoietic progenitor cell lineage selectedfrom the group consisting of granulocyte-macrophage colony-forming cell(GM-CFC), megakaryocyte colony-forming cell (CFC-mega), macrophagecolony-forming cell (M-CFC), granulocyte colony forming cell (G-CFC),burst-forming unit erythroid (BFU-E), colony-forming unit-erythroid(CFU-E), colony-forming cell-basophil (CFC-Bas), colony-formingcell-eosinophil (CFC-Eo), colony-forming cell-megakaryocyte (CFC-Mega),B cell colony-forming cell (B-CFC) and T cell colony-forming cell(T-CFC).
 55. The method of claim 50, further comprising the steps of:(a) contacting a target cell population with at least two concentrationsof a test compound; and (b) calculating the IC50 of the test compound.56. The method of claim 50, further comprising the step of calculatingthe IC90 of the test compound.